Purification and characterization of 3 alpha-hydroxysteroid dehydrogenase from chicken hepatic cytosol.
From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 3 alpha-hydroxysteroid dehydrogenase was purified to an apparently homogeneous state by differential precipitation with ammonium sulfate, followed by column chromatographies with DE 51, DEAE-Toyopearl, and Sephadex G-100. Finally the dehydrogenase was purified 103-fold on the basis of the cytosol fraction. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS) revealed that molecular weight of the purified enzyme was 66 kDa, while that of the native dehydrogenase in the absence of SDS was estimated as 660 kDa or more from the peak of the enzyme in elution profile from Sephacryl S-200 column chromatography. The dehydrogenase required NADPH specifically for reduction of 3-oxo group of 5 beta-androstanedione (Km = 1.6 microM). Optimal temperature for 3-oxo reduction was 50 C in incubation for 10 min.
Morikawa
T
T
Department of Radiopharmacology, Faculty of Pharmaceutical Science, Nagasaki University, Japan.
Amaoki
B
B
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000645:Ammonium Sulfate; D000818:Animals; D002645:Chickens; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005590:Fractional Precipitation; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D013696:Temperature
D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000645:Ammonium Sulfate